Abstract: Process analytical technology (PAT) is gaining more interest in the biomanufacturing industry because of its potential to improve operational control and compliance through real-time quality assurance. Currently, biopharmaceutical producers mainly monitor chromatographic processes with ultraviolet/visible (UV/Vis) absorbance. However, this measurement has a very limited correlation with purity and quantity. The current study aims to determine the concentration of monoclonal antibody (mAb) and host cell proteins (HCPs) using a build-in UV/Vis monitoring during Protein A affinity chromatography and to optimize the separation conditions for high purity of mAb and minimizing the HCPs content. The eluate was analyzed through in-line UV/Vis at 280 and 410 nm, representing mAb and HCPs concentration, respectively. Each 0.1 column volume (CV) fraction of UV/Vis chromatogram peak area were calculated, and different separation conditions were then compared. The optimum conditions of mAb separation were found as 12 CV loading, elution at pH 3.5, and starting the collection at 0.5 CV point, resulting in high mAb recovery of 95.92% and additional removal of 49.98% of HCP comparing with whole elution pool. This study concluded that UV/Vis-based in-line monitoring at 280 and 410 nm showed a high potential to optimize and real-time control Protein A affinity chromatography for mAb purification from HCPs.

Key words: Affinity chromatography, Host cell protein, Monoclonal antibody, Process analytical technology, Spectroscopy

摘要： Process analytical technology (PAT) is gaining more interest in the biomanufacturing industry because of its potential to improve operational control and compliance through real-time quality assurance. Currently, biopharmaceutical producers mainly monitor chromatographic processes with ultraviolet/visible (UV/Vis) absorbance. However, this measurement has a very limited correlation with purity and quantity. The current study aims to determine the concentration of monoclonal antibody (mAb) and host cell proteins (HCPs) using a build-in UV/Vis monitoring during Protein A affinity chromatography and to optimize the separation conditions for high purity of mAb and minimizing the HCPs content. The eluate was analyzed through in-line UV/Vis at 280 and 410 nm, representing mAb and HCPs concentration, respectively. Each 0.1 column volume (CV) fraction of UV/Vis chromatogram peak area were calculated, and different separation conditions were then compared. The optimum conditions of mAb separation were found as 12 CV loading, elution at pH 3.5, and starting the collection at 0.5 CV point, resulting in high mAb recovery of 95.92% and additional removal of 49.98% of HCP comparing with whole elution pool. This study concluded that UV/Vis-based in-line monitoring at 280 and 410 nm showed a high potential to optimize and real-time control Protein A affinity chromatography for mAb purification from HCPs.

关键词: Affinity chromatography, Host cell protein, Monoclonal antibody, Process analytical technology, Spectroscopy